Quality control has long been a necessary and routine procedure in clinical hematology. Accuracy in the counting of various types of blood cells is dependent, in part, upon the use of adequate control products and methods of using the control products. With the numerous types of equipment for particle counting now available, quality control by the use of control products is necessary, since the possibility of an instrument malfunctioning is ever present. The traditional method of maintaining a quality control program for automatic particle counting equipment has consisted of providing fresh human blood as a whole blood standard. However, this fresh blood is usable for only one day, therefore, various manufactured control products which have longer product lifetime have been developed.
Commonly used particles in a control product simulate or approximate the types of particles or cells that are intended to undergo analysis. Consequently, these particles have been frequently referred to as analog particles. The analog particles should be selected or designed so that they have certain characteristics that are similar to those of the particles or cells to be analyzed in the instruments. Exemplary characteristics and parameters include similarities in size, volume, surface characteristics, granularity properties, light scattering properties and fluorescence properties.
Various commercial reference control products are now available, which use various processed or fixed human or animal blood cells as analogs of human blood cells. U.S. Pat. No. 5,512,485 (to Young et al) teaches a hematology control comprising several white blood cell analogs made of processed and fixed animal red blood cells. These fixed red blood cells are fixed at their near native cellular size for simulating specific white blood cell subpopulation in a blood sample. Commercially available hematology controls can also contain red blood cell, platelet, reticulocyte and nucleated red blood cell components.
Nucleated red blood cells (NRBCs), or erythroblasts, are immature red blood cells. They normally occur in the bone marrow but not in peripheral blood. However, in certain diseases such as anemia and leukemia, nucleated red blood cells also occur in peripheral blood. Therefore, it is of clinical importance to measure NRBCs in peripheral blood. In recent years, several detection methods for measuring nucleated red blood cells in a blood sample on a hematology instrument have been reported. U.S. Pat. No. 5,559,037 (to Kim et al.) discloses a method for flow cytometric analysis of nucleated red blood cells and leukocytes. The method uses fluorescence, low angle light scatter and axial light loss measurements to differentiate NRBCs from white blood samples. U.S. Pat. No. 5,879,900 (to Kim et al) further discloses a method of differentiating NRBCs, damaged white blood cells (WBC), WBC and a white blood cell differential in a blood sample by flow cytometry.
U.S. Pat. Nos. 5,874,310 and 5,917,584 (to Li et al) disclose a method of differentiating nucleated red blood cells by measuring two angles of light scatter signals of a blood sample. U.S. Pat. Nos. 5,874,310 and 5,917,584 further disclose a method of differentiating nucleated red blood cells by measuring light scatter and DC impedance signals. U.S. Pat. Nos. 6,410,330 and 6,673,618 (to Li et al) disclose a method of determining NRBC by using DC impedance measurement. U.S. Pat. No. 6,472,215 (to Huo et al) discloses a method of differentiating nucleated red blood cells by an impedance measurement in combination with a three dimensional DC, RF and light scatter measurements.
With the development of the above-referenced detection methods for nucleated red blood cells, several hematology controls containing a nucleated red blood cell component, or NRBC analog, have been reported.
U.S. Pat. Nos. 6,187,590 and 5,858,790 (to Kim et al) disclose a hematology control comprising a nucleated red blood cell (NRBC) analog made of lysed and fixed avian or fish red blood cells, or lysed and fixed human lymphocytes. U.S. Pat. Nos. 6,187,590 and 5,858,790 further disclose the method of preparing the NRBC analog, by lysing avian or fish red blood cells with a lysing reagent for 1 to 5 minutes, followed by fixing nuclei from the cells with a fixative at 60 to 70° C. for up to 10 minutes.
U.S. Pat. Nos. 6,406,915, 6,403,377, 6,399,388, 6,221,668 and 6,200,500 (to Ryan, et al) disclose a hematology control comprising a NRBC analog derived from avian blood cells. The method includes washing avian red blood cells, such as turkey or chicken red blood cells in a buffer solution and fixing the washed cells with glutaraldehyde phosphate solution at room temperature for one day. U.S. Pat. No. 6,448,085 (to Wang et al) discloses a hematology control comprising a nucleated red blood cell (NRBC) analog which is fixed chicken red blood cells obtained from a commercial source.
U.S. Pat. Nos. 6,653,137 and 6,723,563 (to Ryan) disclose methods of making a nucleated red blood cell component for a hematology control by stabilizing blood cells containing a nucleus, or by lysing and removing cytoplasm from blood cells. U.S. Pat. No. 6,723,563 specifically teaches a method of making the nucleated red blood cell component, which comprises the steps of contacting a blood cell which includes a membrane enclosing a nucleus and cytoplasm with a lysing agent for at least 4 hours, removing cytoplasm from within the membrane, but preserving the general structure of the membrane about the nucleus. The method further comprises fixing the blood cells after removing the cytoplasm.
It has been recognized that the nucleated red blood cell analogs produced from different cell sources and processed by different methods can have different properties. For example, the NRBC analog made from lysed and fixed avian blood cells are suitable for fluorescence based measurement methods, but are too small for sizing methods, such as impedance or light scatter measurement methods, for the purpose of simulating human nucleated red blood cells. Furthermore, the nuclei of the stabilized alligator cells without fixation can also be too small when they are analyzed under certain strong lysing conditions used for blood sample analysis on a hematology analyzer. Therefore, it is desirable to have a method of preparing the NRBC analog which enables altering the natural size of cell nucleus of the blood cells used for preparing the analog, to obtain a target size suitable for a predetermined detection domain for simulating human nucleated red blood cells.
In terms of cell property manipulation, U.S. Pat. No. 6,146,901 (to Carver et al) discloses a method for manipulating the optical and electrical properties of a biological particle to achieve selected target values for respective properties. The method includes providing a base biological particle, such as animal red blood cells, having both optical and electrical properties at respective natural values; contacting the particles with a preincubation media which includes a hypotonic buffered solution and a polyhydroxy alcohol to manipulate the optical and electrical properties of the particle; remaining the particle in the preincubation media for an incubation time; subsequently contacting the particle with a primary fixative solution for a period of time. The incubation time and the time in contact with the primary fixative solution are selected to manipulate the respective natural values of the optical and electrical properties of the particle to achieve the target values of the optical and electrical properties. Carver et al's method is used to manipulate the cell properties by leaking a quantity of hemoglobin from the red blood cells without lysing the red blood cells. Carver et al do not teach manipulation or alteration of nucleus properties of a blood cell.